256 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS wide variety of these catalysts available from microbial sources allows the formulator a large degree of freedom in this area. 3. Stability Characteristics of the Enzyme. In addition to the pH-activity characteristics of an enzyme, the formulatot must be cognizant of the pH-stability qualities as well. Selection must be based on a knowledge that the enzyme would be stable in a formulation in the pH range 4.2-5.6. Other factors will be discussed below which contribute to the longevity of an enzyme in a formulation. B. Dosage Form Enzymes can be incorporated in all of the typical dosage forms used by the cosmetic chemist, e.g., ointments, lotions, etc. However, this does not mean that a//enzymes can be incorporated into all existing ointments and lotions. The following are some of the areas of special concern: 1. Enzyme Compatibility. Formulators will immediately recognize the importance of avoiding basic incompatibilities between the enzymes and other components in a formulation. It should be noted, though, that the form an ointment, lotion or emulsion might take could be responsible for the stability characteristics of an enzyme. Many enzymes are susceptible to oxidation, resulting in loss of activity, and most are relatively unstable in solution in an aqueous medium in the absence of stabilizers. It has been our experience that incorporation, as a suspension, in a non- aqueous vehicle provides for excellent product shelf life. The main diffi- culty with this approach is associated with the need for the suspended enzyme to leach out, as a solubilized agent, following application to the skin. If this does not occur, the enzyme will remain suspended, and its activity will not be available. On the other hand, if it leaches out of the oil phase too readily, then the shelf life of the product could suffer. Since the distribution of an ingredient is inherently more uniform when it is in solution rather than suspension, it may be advisable to seek ways to include the enzymes as stable components of the aqueous phase of an emulsion. It is sometimes possible to prepare stable preparations without resorting to the suspension technique by protecting the enzyme in the aqueous phase by such polyhydric compounds as sorbitol or glycerol, and this, in turn, is made the internal phase of a water-in-oil ernulsion. In this way the oil also acts as a protective coating around the enzyme-containing aqueous phase. Of course, it is also possible to incorporate enzymes into a completely nonaqueous ointment. In addition to the drawback that greasy, inelegant cosmetic preparations have, the lack of any moisture in the formulation could hinder or prevent the proper functioning of the enzyme. Unless an extraneous source of moisture is applied, the enzyme must depend on body exudates to supply the necessary water for enzyme solubilization and for

POTENTIAL OF ENZYMES FOR TOPICAL APPLICATION 257 the hydrolyric reactions to take place. This is more likely to occur when the enzyme is applied to a wound which is actively discharging serum, etc. However, for most cosmetic applications, free tissue water will be absent and must therefore be supplied as part of the formulation. 2. Base Compatibility. This refers to the other side of the coin the stability of the emulsion in the presence of the enzymes. One cannot expect to obtain a stable emulsion with a lipid base in the presence of a lipolytic enzyme, nor can one expect a surfactant that is an ester to with- stand the hydrolytic activity of the esterases. So many commercial en- zyme products contain esterase activity present, either as nonspecific contamination or as part and parcel of proteolytic activity, that the selec- tion of the proper emulsifying agent may become a major problem to the formulatot. He must not only be certain that the surfactant is compatible with the enzyme but must be concerned with the reverse condition, as well. In some cases switching from an ester to an ether is all that may be neces- sary, whereas in others the search for the appropriate emulsifier could become a major research project. 3. In Fivo ./lctivity. An ingredient in an ointment can react only at the interface between the skin and the base. One might then expect in vivo enzyme activity to be a function of enzyme concentration up to the point where the interface becomes saturated. In some instances this could be modified by the proper selection of a hydrophilic or lipophilic base. A hydrophilic ointment should serve to attract moisture into a non- aqueous vehicle so that all of the enzyme at the interface would be reactive at any one time. A lipophilic base might be expected to keep at least part of the enzyme inactive throughout a period of contact by preventing the uptake of water. Both types of bases can serve useful purposes, if we assume that a given enzyme would be equally stable in both. If we wanted most rapid action it would appear that the hydrophilic ointment would be more suitable. On the other hand, the lipophilic base would offer a degree of "sustained release" by virtue of the enzyme's inability to hydrate all at once. 4. In Fitro ./lctivity. Usually the activity of a reagent in vitro is not considered if it can be shown to function properly in vivo. In the case of enzymes, however, a label claim can be made for a product only if the active ingredient can be assayed. It has been our experience that the dosage form can be a critical factor in determining whether an enzyme can be quantitatively recovered from a product. More often than not, if the enzyme cannot be recovered under the optimum conditions in our laboratory, it will not be available as an active agent in vivo. As might be expected, vehicles with high Hydrophilic-Lipophilic Balance (HLB) pose the least problem. On the other hand, mixtures that have to